The government has certain rights in the present invention pursuant to NIH grant AI 24639.
1. Field of the Invention
The present invention relates to the field of immune disease treatments and therapeutic agents. The invention also relates to the identification of the human dipeptidyl peptidase-I gene and the preparation of antisense oligonucleotides thereto. Methods of treating immunologically-mediated diseases and malignancies of myeloid cell or cytotoxic lymphocyte origin are also related to the field of the present invention, particularly methods using particular enzyme inhibitors or antisense oligonucleotides specific for inhibiting the expression of human dipeptidyl peptidase-I.
2. Description of the Related Art
Dipeptidyl peptidase-I (DPPI), previously known as cathepsin C, is a lysosomal enzyme that is present in high levels in natural killer cells, cytotoxic lymphocytes, cytotoxic lymphocyte precursors, and myeloid cells. Cytotoxic lymphocytes and myeloid cells have been shown to contain high levels of dipeptidyl peptidase-I (DPPI) within the specialized cytoplasmic granules of these cells..sup.5-7 While DPPI has long been noted to be present at higher levels in spleen and other lymphoid organs as compared to liver or other solid organs,.sup.8 it was only with the elucidation of the role of DPPI in Leu--Leu--OMe-mediated toxicity that this enzyme appeared to be a phenotypic marker of myeloid cells and cytotoxic lymphocytes.
DPPI activity has been examined in populations of purified lymphocytes, myeloid cells and cells of non-bone marrow origin. The amount of DPPI activity in these cell types was found by the present inventors to exceed that of non-cytotoxic lymphocytes, B cells and cells of non-bone marrow origin by up to 20-fold.
DPPI isolated from rat, bovine and porcine tissues has been previously shown to be a lysosomal hydrolase capable of sequentially removing dipeptides from the amino-terminus of suitable substrates..sup.10-13 Highly purified human splenic DPPI has also been shown to demonstrate this substrate specificity. From these reports, the present inventors have determined that a peptide or protein must have an unblocked amino-terminus and the terminal residue cannot be arginine or lysine in order to be a suitable substrate for human DPPI. In addition, the bond to be cleaved must not involve a proline residue. It is clear that with these few limitations, DPPI has a broad substrate range.
DPPI has been characterized as the only known members of the papain enzyme family that exhibits only exopeptidase activity. DPPI has also been characterized as an unusually large molecule, having a molecular weight of 200,000 Da. The other non-DPPI lysosomal thiol proteases are generally monomeric proteins with molecular weights less than 30,000 Da. As a member of the thiol-dependent peptide hydrolases, DPPI is inhibited by general thiol-modifying reagents, such as n-ethylmaleimide, mercurial salts and iodoacetate, by thiol protease inhibitors such as the cystatins and specifically by the active site directed inhibitor, glycyl-phenylalanine diazomethane..sup.17-19
In addition to the above-described hydrolytic activity, these forms of DPPI have also been shown to catalyze the polymerization of dipeptide amides and esters..sup.6,7,9,20-24 This polymerization activity is favored at neutral to alkaline pH, but exhibits a substrate specificity comparable to an acidic hydrolytic activity. The ability of DPPI to polymerize dipeptide esters mediates the toxicity of Leu--Leu--OMe, as this molecule is known to be polymerized to longer peptide chain lengths and is responsible for the toxicity observed with this dipeptide ester within DPPI-enriched cells..sup.6-7 These extended (Leu--Leu).sub.n --OMe polymers, are capable of lysing red blood cells and may have similar effects on the intracellular components of the cells that are sensitive to Leu--Leu--OMe..sup.6,7,9
The role of DPPI in such toxicity was identified by the present inventors through the use of specific inhibitors of DPPI activity, in particular Gly--Phe--CHN.sub.2. While the DPPI dependence of Leu--Leu--OMe toxicity characterized by the present inventors provided the initial motivation for the purification and characterization of this enzyme, the high level of DPPI expression in lymphocytes with cytolytic potential and myeloid cells suggested that an unknown, yet potentially important role for DPPI existed in the function of lymphocytes and myeloid cells. The pattern of DPPI expression and its spectrum of enzymatic activity suggested to the present inventors that DPPI played a role in the post-translational processing and activation of a family of serine proteases expressed only in cells of bone marrow origin.
Lymphocytes with cytolytic potential, natural killer cells, mast cells, and granulocytes express distinct members of a family of serine proteases that have not been identified in other cell types..sup.25-84 These distinct members of the serine protease family of enzymes include the granzymes (found in lymphocytes, natural killer cells), mast cell tryptase and chymase (found in mast cells), leukocyte elastase, cathepsin G, and myeloblastin (found in granulocytes) (Table 1).
TABLE 1 ______________________________________ Serine Proteases of Bone Marrow Derived Cells Protease Cell Type ______________________________________ granzymes cytotoxic T lymphocytes, natural killer cells tryptase mast cells chymase mast cells elastase granulocytes, immature myelomonocytic cells cathepsin G granulocytes, immature myelomonocytic cells myeloblastin granulocytes, immature myeloid cells ______________________________________
While these enzymes share some structural and catalytic features with pancreatic serine proteases, such as trypsin, they have other features that make them unique. The pancreatic serine proteases are stored within the pancreatic exocrine cells in an inactive proenzyme or zymogen form,.sup.49,50 and are synthesized with a typical signal sequence that directs them into lumen of the endoplasmic reticulum. When the signal peptide is cleaved off the nascent protein chain, an "activation" peptide is retained which limits proteolytic activity until the protease is secreted from the cell. The presence of the "activation" peptide is common among the pancreatic proteases. The length of the activation peptide varies from 6 to 20 residues, but always ends with an arginine or lysine..sup.51-53 Therefore, each of the pancreatic proteases can be isolated as a zymogen and activated by treatment with trypsin. The proteases of the complement or clotting cascades are similarly secreted as "inactive" zymogens into the circulation that are activated by cleavage at a specific arginine or lysine (trypsin-sensitive) residue..sup.54
In contrast to the pancreatic serine proteases, the serine proteases of bone marrow cells have only recently been isolated in an inactive zymogen form..sup.55 The inactive zymogen form of these enzymes does not accumulate in cells. Rather, they are transient proteins that limit the activity of the enzyme between the site of synthesis and the cytoplasmic granule, the site where active proteases accumulate. The structure of the synthetic intermediates of these serine proteases was originally deduced from genomic or cDNA cloning studies. All of these serine protease genes contain sequences encoding a typical signal peptide that directs them into the endoplasmic reticulum, followed by a dipeptide predicted to serve as the activation peptide, and then the amino-terminus of the mature enzyme. .sup.25-48 These features are illustrated by the junction sequences of human cathepsin G (HCG), leukocyte elastase (HLE), myeloblastin (MYB), and granzyme A (HGA) (Table 2). The amino-terminus of the active form of each enzyme isolated from cells is underlined. The consensus signal peptidase cleavage site and the cleavage site required to obtain the mature enzyme are marked by arrows.
TABLE 2 __________________________________________________________________________ Amino Acid Sequences Near the Mature N-Termini of Bone Marrow Serine Proteases Signal Sequence Dipeptide N-Terminus Protease -6 -5 -4 -3 -2 -1 1 2 3 4 __________________________________________________________________________ HCG Gly Ala Glu Ala Gly Glu Ile Ile Gly Gly HLE Thr Ala Leu Ala Ser Glu Ile Val Gly Gly MYB Ala Ala Arg Ala Ala Glu Ile Val Gly Gly HGA Glu Asp Val Cys Glu Lys Ile Ile Gly Gly .uparw. .uparw. __________________________________________________________________________
It is clear from the sequences shown above that, with the exception of granzyme A, the processing of these serine protease enzymes could not be carried out by a trypsin-like protease. Therefore, a distinct mechanism must exist for the activation of the serine proteases. Inspection of the deduced amino acid sequences of the serine proteases associated with bone marrow derived cells suggested to the inventors that in each case, the dipeptide "activation" peptide would be a suitable substrate for DPPI.
Salvesen and Enghild demonstrated the transient presence of a dipeptide-bearing form of elastase and cathepsin G in U-937 cells.sup.80. This form was judged not to be catalytically active based on failure to interact with an affinity matrix, aprotinin-agarose. However, there was no direct measurement of hydrolysis of specific peptide or protein substrates. In addition, the enzyme responsible for the dipeptide processing and activation of these serine proteases has not been identified. The identification of the enzyme responsible for the processing/activation of serine proteases was, however, believed to be important to characterizing and controlling the cell-mediated cytotoxicity caused by cell types found by the present inventors to have high concentrations of these enzymes.
Cell mediated cytotoxicity has been shown to be a major component of immune responses directed against allogeneic tissues..sup.3,6 Defining the role of cytotoxic lymphocytes during evolution of alloimmune responses is made difficult in part because of the heterogeneity of functions mediated by phenotypically similar lymphocytes. Whereas studies of both human and murine T-cells have indicated that the bulk of T cytokine production and helper function is mediated by CD4(+) T-cells, and as the majority of cytotoxic effector cells are CD8(+), a number of exceptions to this generalization have been noted. For example, in alloimmune responses, both T-cytokine and T-cytotoxic responses directed against Class II MHC differences have been shown to be mediated by CD4(+)T-cells, whereas isolated Class I MHC differences stimulate cytokine and cytotoxic responses from CD8(+)T-cells..sup.9,10 Therefore, expression of CD8 or CD4 cannot be used to identify T-cell subsets which specifically manifest cytotoxic activity..sup.9-11
Approaches based on differential screening of cDNA libraries have identified "CTL specific" serine proteases including granzymes A-G in the mouse and granzyme A and granzyme B in the human..sup.12-18 Transcription of genes for these proteases precedes acquisition of cytolytic effector function during primary in vitro mixed lymphocyte cultures (MLC)..sup.19
The present Inventors postulate a role for effector cells expressing the granzyme A gene in allograft rejection. However, a specific role for granzymes in CTL effector function has been difficult to demonstrate directly. For example, whereas inhibitors of granzyme A or B activity have been shown to impair CTL lytic function, virtually all such inhibitors act on broad classes of serine proteases..sup.23 Thus, it is not clear that granzymes are the sole target of these inhibitors. Although purified granzymes have not been demonstrated to have direct lytic activity, granzyme A may play other roles, such as in CTL-mediated degradation of target cell DNA,.sup.24 or in modifying the activity of other granule associated effector molecules..sup.25 Granzyme A gene expression in infiltrating lymphocytes has already been reported to be a useful marker of human cardiac rejection..sup.22 Granzymes may also play a role in mediating rapid detachment of activated lymphocytes from targets..sup.26 Granzyme A may be a growth factor for B or T-cells.sup.27,28 or may be active in the degradation of extracellular matrix proteins..sup.29 Thus, while it is clear that granzymes are expressed uniquely by cells with cytotoxic potential, their precise role in cytolysis remains to be identified.
Target cell injury induced by CTL is characterized by a pattern of early nuclear condensation and DNA fragmentation that is not observed following complement-mediated lysis..sup.39 While perforin containing granules and chromatographically purified perforin have been reported in one study to induce DNA degradation,.sup.40 other investigators have not been able to induce target cell DNA fragmentation even with highly purified perforin preparations..sup.41 A number of investigators have suggested that CTL mediated lysis may involve multiple mechanisms..sup.10 Moreover, while granzyme and perforin expressing granular effector cells appear to be the predominate mediators of spontaneous natural killer cell function and alloantigen-specific CTL function generated in primary in vitro MLC, agranular allospecific CTL without detectable granzyme A or perforin activity have been reported to be induced following in vivo intraperitoneal priming with allogeneic cells..sup.43
It appeared to the present inventors that use of specific inhibitors of DPPI enzymatic activity of or the development of a specific antisense oligonucleotide directed against the expression of DPPI would provide a selective and potent means of controlling the processing of the serine proteases, and thereby the cellular damage induced thereby, in pathologies which involved the activity of cells of bone marrow origin. Such was supported by the observations of the inventors that DPPI was important to the function of several types of cell damage mediated by lymphocytes, or by myeloid cells via granule serine protease mediated mechanisms.
It appeared to the present inventors that specific forms of antisense therapy directed toward inhibiting the expression of the human DPPI gene or the human DPPI protein would provide a potential therapy for the treatment of many human diseases linked to the action or malfunction of cytotoxic lymphocytes on cells of myeloid lineage, such as leukemia, GVHD, graft rejection, and the like. However, neither the protein or the gene for human DPPI had been characterized. Only recently has the sequence for the rat DPPI protein and rat cDNA clone been reported.sup.79. However, prior to purification of human DPPI, the human DPPI protein, which had not yet been characterized, could not be used to determine if any homology with the rat DPPI protein existed. Therefore, significant technical difficulties remained in both isolating the human protein and isolating the human gene which encodes the protein, before the types of specific oligonucleotide DPPI inhibitors contemplated by the inventors could be synthesized.
In light of the present inventors' work regarding the important role of DPPI in the activation of cells of bone marrow origin and the role of DPPI dependent effector on mechanisms in several human pathologies, the development of particular antisense oligonucleotides specific for binding the human DPPI gene and/or RNA would provide an important therapeutic tool in the treatment of immunologically related diseases.